Fish Gut Microbiota Research Articles

Changes in intestinal morphology and microbiota caused by dietary administration of inulin and Bacillus subtilis in gilthead sea bream (Sparus aurata L.) specimens

Changes produced in gilthead sea bream (Sparus aurata L.) intestinal morphology and microbiota caused by dietary administration of inulin and Bacillus subtilis have been studied. Gilthead sea bream specimens were fed diets containing 0 (control), inulin (10 g kg−1), B. subtilis (107 cfu g−1), or B. subtilis + inulin (107 cfu g−1 + 10 g kg−1) for four weeks. Curiously, fish fed the experimental diets (inulin, B. subtilis, or B. subtilis + inulin) showed the same morphological alterations when studied by light and electron microscopy, while significant differences in the signs of intestinal damage were detected by the morphometric study. All of the observed alterations were present only in the gut mucosa, and intestinal morphometric study revealed no effect of inulin or B. subtilis on the intestinal absorptive area. Furthermore, experimental diets cause important alterations in the intestinal microbiota by significantly decreasing bacterial diversity, as demonstrated by the specific richness, Shannon, and range-weighted richness indices. The observed alterations demonstrate that fish fed experimental diets had different signs of gut oedema and inflammation that could compromise their body homeostasis, which is mainly maintained by the epithelial lining of the gastrointestinal tract. To our knowledge, this is the first in vivo study regarding the implications of the use of synbiotics (conjunction of probiotics and prebiotics) on fish gut morphology and microbiota.

Effects of dietary 60 g kg−1dried distiller's grains in least-cost practical diets on production and gut allochthonous bacterial composition of cage-cultured fish: comparison among fish species with different natural food habits

Dried distiller's grain (DDG) is considered as an alternative ingredient of dietary feed due to its high contents of protein, fibre and fat. In this study, 60 g kg-1 of DDG was used to feed grass carp (Ctenopharyngodon idella), bluntnose black bream (Megalobrama amblycephala), gibel carp (Carassius gibelio) and black carp (Mylopharyngodon piceus) for 8 weeks, and its effect on fish production and gut allochthonous microbiota was investigated for the development of a suitable fish feed high in nutrients and low in cost for polyculture freshwater fish. DDG supplementation resulted in the less weight gain and higher feed conversion ratio of black carp (P < 0.05), but had no significant effects on other fish or parameters. PCR–denaturing gradient gel electrophoresis (DGGE) analysis indicated that all four fish species had some common and unique bacteria in their digestive tracts, and the gut microbiota of bluntnose black and gibel carp fed the control diet and DDG diets were very similar (Cs > 91%); of them, the total counts of intestinal bacteria studied by qPCR increased in grass carp (P < 0.05) and depressed in black carp (P < 0.05) when fed dietary DDG. Thus, we assumed that dietary DDG modulated production and gut microbiota of fish in a host-specific way.

Evaluation of the presence and efficiency of potential probiotic bacteria in the gut of tilapia (Oreochromis niloticus) using the fluorescent in situ hybridization technique

The Fluorescent in situ Hybridization (FISH) technique was employed to enumerate potential probiotic and putative pathogenic bacteria in the gut of tilapia (Oreochromis niloticus). Bacteria used in the study were isolated from water, sediment and intestines of tilapia (Oreochromis niloticus) raised in an aquaculture system. These isolates were tested in vitro on antagonism tests against putative pathogenic bacteria (Aeromonas hydrofila, Enterococcus faecalis, Edwardsiella tarda, Pseudomonas fluorescens and Pseudomonas putida), also isolated from the same aquaculture system. Two isolates that inhibited largest number of pathogenic bacteria were identified by sequencing as Bacillus sp. and Enterococcus sp. and were added to the commercial feed (106cellsg−1) for in vivo tests. Treatments of the in vivo experiment were: 1) Control — fish fed with no added bacteria, 2) Bacil. — fish fed diets containing Bacillus sp.; 3) Enter. — fish fed diets containing Enterococcus sp., and 4) Bacil.+Enter. — fish fed diets containing Bacillus sp. and Enterococcus sp. (1:1). Each treatment consisted of four replicates with 15 juveniles of tilapia (O. niloticus — 16.74±4.35g e 9.82±0.85cm). The experiment lasted for 30days and at the end of this period, three fish from each tank were killed, and the intestines were taken for microbiological analysis by FISH technique, where Bacillus and Enterococcus, as well as two putative pathogenic bacteria (Aeromonas and Pseudomonas sp.) were quantified. Enterococcus sp. and Bacillus sp. were present in high number in the gut microbiota of fish. However, Bacillus sp., showed an increase in its abundance, indicating a successful incorporation of this potential probiotic bacteria into the tilapia gut microbiota. Furthermore, in the Bacil. treatment it was observed a significant reduction of Aeromonas and Pseudomonas sp. abundances compared with the other treatments. These results indicate that the FISH technique is a potential tool to characterize the dynamics of potential probiotic bacteria and their efficiency in the control of pathogenic bacteria.

Characterization of enzyme-producing bacteria isolated from the gut of Asian seabass,Lates calcariferand milkfish,Chanos chanosand their application for nutrient enrichment of feed ingredients

Aquatic bacteria influence the composition of gut microbiota in fish (Cahill 1990). Like all vertebrates and invertebrates, fish also harbour microbial populations in their digestive tracts (Trust & Sparrow 1974). In all higher vertebrates food gets digested by the action of enzyme secreted by host and also enzyme produced by the intestinal microbiota (Kar, Roy, Sen & Ghosh 2008). Endogenous digestive enzyme has been studied by several workers in freshwater fish (Dhage 1968; Das & Tripathi 1991). Few reports are also available with regard to enzyme-producing gut bacteria of freshwater fish (Saha & Ray 1998; Ghosh, Sen & Ray 2002; Kar & Ghosh 2008; Kar et al. 2008; Askarian, Zhou, Olsen, Sperstad & Ring 2012; Khan & Ghosh 2012). Different gut microbes having considerable cellulase, amylase and protease activity had been isolated from freshwater fish (Bairagi, Sarkar, Sen & Ray 2004; Saha, Roy, Sen & Roy 2006; Mondal, Roy, Sen & Ray 2008). Gatesoupe, Zambonino Infante, Chu and Quazuguel (1997) reported amylase-producing Vibrio spp. isolated from seabass (Dicentrarchus labrax) larvae. They, however, did not specify the exact site of the gut (proximal, mid or distal intestine) where the population of these cellulaseand amylase-producing microbes and the enzyme activity is maximum. Reports on the existence of cellulase activity in the digestive system of fish are with contradictory results (Ray, Ghosh & Ring 2012). The ruminants can digest cellulose through the action of cellulose secreted by the anaerobic gut fungi and bacteria present in rumen. There is ongoing interest in screening culturable cellulase-producing microorganisms from different sources. The major aim of this study was to investigate the autochthonous cellulase-producing bacteria in the gastrointestinal tracts of two brackishwater fish namely Asian seabass and milkfish and they were used for nutrient enrichment of the feed ingredients through solidstate fermentation. The adult brackishwater fish 6 nos. of each species) were collected from brackishwater tide fed farm of Kakdwip (Lat. 2151′ 15.01′′–2151′30.77′′N, Long. 8810′58.44′′– 8811′12.09′′E), South 24 Parganas, West Bengal, India. Fish were starved for 48 h to clear their alimentary tracts before dissection (Bairagi, Sarkar Ghosh, Sen & Ray 2002a; Kar et al. 2008). During the sampling periods the water temperature varied between 30°C and 35°C. The average weight, length of fish and average weight of digestive tract were recorded (Table 1). Fish was thoroughly scrubbed with 1% iodine solution after sacrifice (Trust & Sparrow 1974). After dissection on ice the whole alimentary tract was removed and cleaned with chilled sterile physiological saline (0.9% NaCl in PBS buffer, pH 7.2). Subsequently, the pieces of digestive tract were

Intestinal microbiota composition in fishes is influenced by host ecology and environment

The digestive tracts of vertebrates are colonized by complex assemblages of micro-organisms, collectively called the gut microbiota. Recent studies have revealed important contributions of gut microbiota to vertebrate health and disease, stimulating intense interest in understanding how gut microbial communities are assembled and how they impact host fitness (Sekirov et al. 2010). Although all vertebrates harbour a gut microbiota, current information on microbiota composition and function has been derived primarily from mammals. Comparisons of different mammalian species have revealed intriguing associations between gut microbiota composition and host diet, anatomy and phylogeny (Ley et al. 2008b). However, mammals constitute <10% of all vertebrate species, and it remains unclear whether similar associations exist in more diverse and ancient vertebrate lineages such as fish. In this issue, Sullam et al. (2012) make an important contribution toward identifying factors determining gut microbiota composition in fishes. The authors conducted a detailed meta-analysis of 25 bacterial 16S rRNA gene sequence libraries derived from the intestines of different fish species. To provide a broader context for their analysis, they compared these data sets to a large collection of 16S rRNA gene sequence data sets from diverse free-living and host-associated bacterial communities. Their results suggest that variation in gut microbiota composition in fishes is strongly correlated with species habitat salinity, trophic level and possibly taxonomy. Comparison of data sets from fish intestines and other environments revealed that fish gut microbiota compositions are often similar to those of other animals and contain relatively few free-living environmental bacteria. These results suggest that the gut microbiota composition of fishes is not a simple reflection of the micro-organisms in their local habitat but may result from host-specific selective pressures within the gut (Bevins & Salzman 2011).

Enzyme-producing bacteria isolated from fish gut: a review

Digestion of food depends on three main factors: (i) the ingested food and the extent to which the food is susceptible to the effects of digestive enzymes, (ii) the activity of the digestive enzymes and (iii) the length of time the food is exposed to the action of the digestive enzymes. Each of these factors is affected by a multitude of secondary factors. The present review highlights the experimental results on the secondary factor, enzymatic activity and possible contribution of the fish gut microbiota in nutrition. It has been suggested that fish gut microbiota might have positive effects to the digestive processes of fish, and these studies have isolated and identified the enzyme-producing microbiota. In addition to Bacillus genera, Enterobacteriaceae and Acinetobacter, Aeromonas, Flavobacterium, Photobacterium, Pseudomonas, Vibrio, Microbacterium, Micrococcus, Staphylococcus, unidentified anaerobes and yeast are also suggested to be possible contributors. However, in contrast to endothermic animals, it is difficult to conclude the exact contribution of the gastrointestinal microbiota because of the complexity and variable ecology of the digestive tract of different fish species, the presence of stomach and pyloric caeca and the relative intestinal length. The present review will critically evaluate the results to establish whether or not intestinal microbiota do contribute to fish nutrition.

Use of chitin and krill in aquaculture - the effect on gut microbiota and the immune system: a review

Chitin consists of β-1,4-linked N-acetylglucosamine residues and is estimated as the second most abundant biomass in the world after cellulose. However, relatively little chitin is utilized as a material for industrial, agricultural and medical applications and aquacultural purposes. Chitin may be useful as a constitutive material in formulated fish feed, and the interesting effects in fish merit further evaluation. There is evidence that fish and aquatic animals harbour a gut bacterial community that is distinctly different from that reported in the surrounding habitat or in the diet. Thus, the gut environment provides a specific niche, and bacterial activity in the gut is not merely a continuum of that observed in the environment. Today, it is well accepted that the gut microbiota in fish are modulated by dietary manipulations. But to what extent can dietary chitin and krill (chitin-rich) modulate the intestinal microbiota of fish and how do these dietary components affect the immune system? These questions will be discussed in the present review.

Do stocking densities affect the gut microbiota of gibel carp Carassius auratus gibelio cultured in ponds?

The aim of the present study was to evaluate the intestinal microbial communities of gibel carp (Carassius auratus gibelio) cultivated in two beach ponds at different stocking densities. The two ponds were both ~3.33 hm 2 in acreage and ~1.5 m in depth. The stocking densities included one intensive with 2 fish m –3 while the other treated as semi-intensive with 1 fish m –3 . The gut microbiota (both allochthonous and autochthonous) were sampled after 135 days of feeding. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene segments was used to evaluate the bacterial community. Actinobacteria, Cyanobacteria, Firmicutes, Fusobacteria, Proteobacteria and some unclassified_bacteria taxa were identified in gut samples and feed. Similar bacterial communities (C s=0.83) were observed with respect to the autochthonous and allochthonous gut microbiota of gibel carp cultured in the intensive culture pond. In contrast to these results, some difference (C s =0.61) was observed in the gut microbiota of fish reared in the semi-intensive culture pond. Our results indicated that the difference in the bacterial communities between allochthonous bacteria and gut associated bacteria of gibel carp was not constant and was modulated by the stocking density.

Gut microbiota of fast and slow growing grouper Epinephelus coioides

Although the gut microbiota of fish has been studied extensively using traditional or molecular techniques, little information is available on the correlation between gut microbiota and growth of host fish. In the present study, the gut microbiota of two groups of juvenile grouper Epinephelus coioides, slow growing (SG) and fast growing (FG) grouper at 75 days post-hatch (DPH), were investigated by using standard isolation and characterization procedures. The results showed that 4 Vibrio specieswere isolated and comprised 12.3% of the total gut bacteria in SG grouper, whereas only two Vibrio species were isolated and comprised 3.6% of the total bacteria in FG grouper. At the same time, Bacillus pumilus, Bacillus clausii and Psychroacter sp. were only isolated and dominated in the gut of FG fish. The 3 species showed antagonistic effect on pathogenic Vibrio, this may cause the lower number and less species of Vibrio in the gut of FG grouper and suggesting fast growing fish might harbor a favorable microbiota. The number of total cultivable aerobic and facultative anaerobic bacteria was similar in the gut of two groups, 5.4×106 CFU/g and 9.0×106CFU/g in SG and FG group, respectively. Besides those most common bacteria such as Vibrio spp., Pseudomonas spp. and Acinetobacter spp., several Gram-positive and Gram-negative bacteria not normally in the gut of marine fish were also isolated from both groups of fish. The bacteria in the gut of grouper could be classified into three groups belonging to γ-proteobacteria, β-proteobacteria and Bacilli class. Key words: Gut microbiota, fast growing grouper, slow growing grouper,Epinephelus coioides

A review on the interactions between gut microbiota and innate immunity of fish: Table 1

Although fish immunology has progressed in the last few years, the contribution of the normal endogenous microbiota to the overall health status has been so far underestimated. In this context, the establishment of a normal or protective microbiota constitutes a key component to maintain good health, through competitive exclusion mechanisms, and has implications for the development and maturation of the immune system. The normal microbiota influences the innate immune system, which is of vital importance for the disease resistance of fish and is divided into physical barriers, humoral and cellular components. Innate humoral parameters include antimicrobial peptides, lysozyme, complement components, transferrin, pentraxins, lectins, antiproteases and natural antibodies, whereas nonspecific cytotoxic cells and phagocytes (monocytes/macrophages and neutrophils) constitute innate cellular immune effectors. Cytokines are an integral component of the adaptive and innate immune response, particularly IL-1 beta, interferon, tumor necrosis factor-alpha, transforming growth factor-beta and several chemokines regulate innate immunity. This review covers the innate immune mechanisms of protection against pathogens, in relation with the installation and composition of the normal endogenous microbiota in fish and its role on health. Knowledge of such interaction may offer novel and useful means designing adequate therapeutic strategies for disease prevention and treatment.

Characterisation of the microbiota associated with intestine of Atlantic cod ( Gadus morhua L.) : The effect of fish meal, standard soybean meal and a bioprocessed soybean meal

Populations of aerobic heterotrophic bacteria present in the gastrointestinal (GI) tract of healthy Atlantic cod ( Gadus morhua L.) fed three different diets, fish meal, standard or a bioprocessed soybean meal (BPSBM), were estimated using the dilution plate technique. A total of 944 isolates were characterised by biochemical and physiological properties and 425 isolates were identified further by sequencing the 16S rRNA genes. Our results showed that gut microbiota were affected by dietary manipulation. The GI tract of fish fed fish meal was dominated by Gram-positive bacteria of the genera Brochothrix and Carnobacterium. The Gram-negative bacteria Chryseobacterium spp. and Psychrobacter glacincola, and Gram-positive bacteria belonging to Carnobacterium, dominated in the digestive tract of fish fed soybean meal. In contrast to these results, genus Psychrobacter dominated in the GI tract when fish were fed BPSBM. Until recently, it was generally suggested that the gut microbiota of fish were less diverse than in homoeothermic animals. However, the present study identified several “new” bacterial species isolated from the alimentary tract of Atlantic cod. These “new” bacterial species are not normally isolated from the GI tract of fish. Based on our finding we suggest that the GI tract microbiota of fish might not be as simple as believed. Antagonistic activity of carnobacteria regarding inhibition of growth of two fish pathogens ( Aeromonas salmonicida ssp. salmonicida and Vibrio anguillarum) was observed. However, some difference in the antibacterial activity of Carnobacterium spp. was observed. Whether this antagonistic activity has any effect in challenge studies will be discussed, especially in relation to the finding that the digestive tract is one of the major infection routes in fish.